Doctoral Dissertations

Date of Award

8-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

John W. Koontz

Committee Members

Jerry P. W.

Abstract

Tyrosine aminotransferase (TAT) is a liver-specific enzyme that is regulated by glucocorticoids, agents that increase the intracellular concentration of cyclic AMP, and insulin. Glucocorticoids and cyclic AMP analogs increase TAT activity through transcriptional activation of the TAT gene. This same result has been demonstrated for insulin in adult rat liver and in Fao rat hepatoma cells. However, insulin will inhibit TAT activity in the fetal rat liver, and will inhibit TAT transcription in the neonatal rat liver. Previous work in this laboratory has shown that insulin will inhibit TAT transcription in the KRC-7 clone of H35 rat hepatoma cells, while at the same time causes an increase in TAT activity. This suggests that the KRC-7 clone represents a neonatal rat liver with respect to regulation of TAT. To test the hypothesis that insulin stabilizes TAT mRNA to contribute to the increase in activity, half-life determinations of TAT mRNA were made in the presence and absence of insulin. Although no effect of insulin on the half-life of TAT mRNA was observed, it was later shown that insulin changes the degradation rate of TAT (protein) which may account for the observed increase in enzyme activity after insulin treatment.

Our laboratory is interested in studying the regulation of TAT by insulin at the molecular level. Subclones of the TAT 5' flanking region have been constructed and the sequence of the TAT 5' flanking region has been determined to -1300 bp. A gel-shift assay has been used to study the DNA-protein interactions in the flanking region. The ability to detect DNA-protein interactions correlates well with regions of known DNase I hypersensitivity. By analysis of the gel-shift patterns obtained from different regions the proteins that interact with the TAT 5' flanking region are found in non-hepatic cells. In addition, no differences in these interactions were observed after hormonal treatment.

Since TAT is expressed exclusively in the liver, it was of interest to determine if a known liver-specific trans-acting factor, APF, could be one of the protein(s) that is responsible for the tissue-specific expression of TAT. Although there are two APF consensus binding sites in the 5' flanking region of the TAT gene, competition analysis demonstrated that APF does not interact with these consensus sites. Furthermore, KRC-7 cells express the adult form of APF, and not the variant form found in dedifferentiated hepatoma cells. These results are discussed.

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